Modulation of pulmonary desmosomes by inhaler therapy in preterm-born children with bronchopulmonary dysplasia

Despite evidence demonstrating persistent lung function deficits in preterm-born children, especially in those who had bronchopulmonary dysplasia (BPD) in infancy, the underlying biological mechanisms explaining these lung function deficits remain poorly understood. We characterised the exhaled breath condensate (EBC) proteome in preterm-born children, with and without BPD; and before and after inhaler treatment. EBC from children aged 7–12 years, from the Respiratory Health Outcomes in Neonates (RHiNO) study, were analysed by Nano-LC Mass Spectrometry with Tandem Mass Tag labelling. Children with percent predicted forced expiratory volume in 1 second ≤ 85% were enrolled to a 12-week blinded randomised trial of inhaled corticosteroids alone (ICS) or with long-acting β2-agonist (ICS/LABA) or placebo. EBC was analysed from 218 children at baseline, and 46 children received randomised inhaled therapy. 210 proteins were detected in total. For the 19 proteins present in every sample, the desmosome proteins: desmoglein-1, desmocollin-1 and plakoglobin were significantly decreased, and cytokeratin-6A was increased in preterm-born children with BPD when compared to preterm- and term-born controls. ICS/LABA treatment significantly increased abundance of desmoglein-1, desmocollin-1 and plakoglobin in the BPD group with low lung function, and significantly increased plakoglobin in those without BPD. No differences were noted after ICS treatment. Exploratory analyses of proteins not detected in all samples suggested decreased abundance of several antiproteases. This study provides proteomic evidence of ongoing pulmonary structural changes with decreased desmosomes in school-aged preterm-born children with BPD and low lung function, which was reversed with combined inhaled corticosteroids and long-acting β2-agonists therapy.

cartridge according to the manufacturer's instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).
All spectra were acquired using an Orbitrap Fusion Lumos mass spectrometer controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with an automatic gain control (AGC) target of 200,000 and a max injection time of 50ms.
Precursors were filtered with an intensity threshold of 5000, according to charge state (to include charge states 2-7) and with monoisotopic peak determination set to Peptide.
The MS2 precursors were isolated with a quadrupole isolation window of 0.7m/z. ITMS2 spectra were collected with an AGC target of 10 000, max injection time of 70ms and CID collision energy of 35%.
For FTMS3 analysis, the Orbitrap was operated at 50,000 resolution with an AGC target of 50,000 and a max injection time of 105ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalised collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous Precursor Selection (SPS) was enabled to include up to 5 MS2 fragment ions in the FTMS3 scan.

Data Analysis
The raw data files were processed and quantified using Proteome Discoverer software v2.1 (Thermo Scientific) and searched against the UniProt Human database (downloaded October 2019: 150786 entries) using the SEQUEST HT algorithm. Peptide precursor mass tolerance was set at 10ppm, and MS/MS tolerance was set at 0.6Da. Search criteria included oxidation of methionine (+15.995Da), acetylation of the protein N-terminus (+42.011Da) and Methionine loss plus acetylation of the protein N-terminus (-89.03Da) as variable modifications and carbamidomethylation of cysteine (+57.021Da) and the addition of the TMT mass tag (+229.163Da) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of 2 missed cleavages were allowed. The reverse database search option was enabled, and all data was filtered to satisfy false discovery rate (FDR) of 5%.    Figure 4: Protein network map of significant protein differences before and after ICS/LABA treatment.